Diamine Oxidase (E.C.1.4.3.6) (hereinafter called DAO) catalyses the oxidation of diamines such as histamine, putrescine and cadaverine, which yields an aminoaldehyde. During early investigations the enzyme was believed to only oxidise histamine: thus the enzyme was initially named Histaminase by Best and McHenry (1929). Further investigations proved that the enzyme was capable of oxidation of other diamines such as those listed above. The nomenclature currently in use, Diamine Oxidase, was later suggested by Zeller (1965).
Until 1965, the only intracellular mammalian diamine oxidase that had been purified was pig kidney histaminase (Kapeller-Adler, 1963; and Mondovi, 1964). Considerable purification of pig kidney histaminase (200- to 240-fold) was obtained by Tabor in 1951. Higher degrees of purification were successively obtained by applying column chromatography and electrophoresis (Mondovi, 1967a; and Uspenskaia, 1958). Diamine oxidase has also been purified from the pig kidney cortex by chromatography on hydroxylapatite and DEAE-Sephadex A-50, and gel filtration through Sephadex G-200 (Bardsley, 1971). This gave an enzyme preparation purified 230-fold with a 35% yield.
AH-Sepharose 4B was used as an affinity sorbent after partial purification of the pig kidney diamine oxidase by means of controlled heating, fractionation with ammonium sulphate and precipitation at pH 5.3. The AH-Sepharose adsorbed inactive proteins in preference to the diamine oxidase. This method provided an enzyme purified 670-fold, but with only a 14% yield (Floris, 1976). Increased yields of the enzyme from pig kidney was achieved by altering the heating conditions used by Floris (1976) on the homogenate, followed by ammonium sulphate fractionation and column chromatography on DEAE-Cellulose. Dialysis of the enzyme against 0.005M phosphate buffer resulted in an electrophoretically homogenous enzyme preparation purified 2400-fold with a 60% yield (Kluetz, 1977b).
Another method developed for purifying diamine oxidase from pig kidney cortex avoided heating the homogenate. The extract obtained after ammonium sulphate fractionation and centrifugation, was subjected consecutively to column chromatography on DEAE-Sephadex A-50 and hydroxylapatite, with subsequent gel filtration through Sephadex G-150. The enzyme was purified 1350-fold with a 20% yield, and was shown to be homogeneous by SDS-PAGE (Yamada, 1967). To simply the purification of pig kidney diamine oxidase, an affinity sorbent DAH-Sepharose was introduced by Klimova (1976). An extract of pig kidney cortex homogenate obtained after centrifugation of the ammonium sulphate treated sample was chromatographed on DAH-Sepharose and gave an enzyme preparation purified 200-fold with a 20% yield.
Human kidney diamine oxidase was purified 1800-fold by heating the homogenate, treating it with a mixture of ethanol and chloroform, chromatography on CM-Sephadex, and followed by fractionation with ammonium sulphate and gel filtration through Sephadex G-200 (Shindler, 1976).
Preparation of diamine oxidase from human placenta was achieved by fractionation with ammonium sulphate; chromatography on DEAE-Cellulose and phosphocellulose, and gel filtration through Sephadex G-200 (Smith, 1967). This preparation of diamine oxidase was purified 515-fold, although it still contained haptoglobin and methemoglobin. Paolucci (1971) obtained a 9600-fold purification of placental diamine oxidase with an 8% yield. The proteins of the human placenta were extracted using an acetone powder technique and precipitated with ethanol absorbed on DEAE-Cellulose; fractionated with ammonium sulphate, chromatographed on DEAE-Collulose and subjected to gel filtration through Biogel A-5m. A 4600-fold purification of human placental diamine oxidase was obtained by Bardsley (1974). The methods used in the purification of the enzyme were as those reported for Smith (1967), although a final chromatography step on DEAE-Sephadex or hydroxylapatite was employed which resulted in an increase in specific activity and gave an enzyme which was homogeneous by SDS-PAGE.
Hata (1976) obtained a 1200-fold purification of human placental diamine oxidase ontained by the method described by Bardsley (1974). The polyacrylamide gel electrophoresis pattern of purified human placental diamine oxidase gave a broad band, with the preparation not purified to the same extent as that described by Bardsley (1974). The purification of human placental diamine oxidase was also described by Crabbe (1976), using a modification of the procedure described by Bardsley (1974). Column chromatography incorporated DEAE-Sephadex with ionic strength and pH-gradient elution, together with affinity chromatography on Concavalin A-Sepharose. This gave a 30,800-fold purification with a 33% yield. Lin (1981) purified placental diamine oxidase in what was referred to as a one-step purification. In this procedure, placentae were homogenized and centrifuged, with the resultant supernatant applied to the affinity gel, cadaverine AH-Sepharose as was previously employed by Baylin (1975b). Elution of the enzyme was accomplished using chromotropic acid, and provided a 1800-fold purification of the enzyme which was homogeneous by SDS-gel electrophoresis.
Plasma diamine oxidase from women in the third trimester of pregnancy was purified 3000-fold with a 25% yield by means of an affinity sorbent sepharose, covalently linked with cadaverine (Baylin, 1975b). The enzyme was eluted from the adsorbent with heparin; electrophoresis by SDS-PAGE presented two protein fractions, one of which displayed the diamine oxidase activity.
Diamine oxidase was isolated from human female amniotic fluid by chromatography on DEAE-Sephadex and affinity chromatography on cadaverine-Sepharose, which was shown by SDS-PAGE to contain only 10% inactive protein (Tufvesson, 1978a). Diamine oxidase from human male serum previously given intravenous heparin (10000 I.U.) one hour before collecting the blood, was isolated by cadaverine-Sepharose affinity chromatography and then by chromatography on DEAE-Cellulose (Tufvesson, 1978b).
The enzyme can also be found in kidney and other organs in a variety of animal species. A great deal of time had been devoted to characterization studies of pig kidney (Kapeller-Adler & Macfarlane (1963)) and pig serum (Blaschko, Friedman, et al (1959)) DAO, but the human enzyme (Crabbe, (1979)) has been only poorly characterised.
According to previous studies, there seemed to be several forms of DAO and these forms exhibited some tissue specificity. Although presence of the enzyme in human placenta and human pregnancy plasma was confirmed by Swanberg (1950), this group was unable to establish any difference between the forms of human placental and pregnancy plasma DAO. Moreover, Tufvesson (1978) could find no difference between amniotic fluid DAO and pregnancy serum DAO, although he suggested that heparin stimulated male serum DAO was different.
Four active protein fractions, with molecular weights of 125,000, 250,000, 375,000 and 500,000 daltons were found in preparations of human placental diamine oxidase by Paolucci (1971). The preparation of human placental diamine oxidase obtained by Hata (1976) was purified 1200-fold, and the presence of a broad band on polyacrylamide gel electrophoresis indicated the presence of a contaminant protein which was demonstrated distinctly by immunoelectrophoresis and SDS-PAGE. The molecular weight was calculated to be approximately 300,000 by comparison with a calibration curve by gel filtration and SDS-PAGE. The molecular weight of the subunit was 170,000, which is about two times of 90,000 obtained by Bardsley (1974) and Lin (1981).
By the use of gel filtration through Sephadex G-200, polyacrylamide gel electrophoresis in the presence of SDS, or ultracentrifugation, it was demonstrated that a monomeric, catalytically active form of human placental diamine oxidase had a subunit molecular weight of 70,000 (Crabbe, 1976). This value is close to that reported by Baylin (1975b) for diamine oxidase from pregnancy plasma, which would appear to be a monomer. Sedimentation-equilibrium ultracentrifugation results indicated a single species with a molecular weight of 235,000, which could be explained on the basis of concentration-dependant-aggregation, to be dimeric and tetrameric species which were in equilibrium with each other (Crabbe 1976).
A protein component with a molecular weight of 185,000 was detected by means of gel filtration through Sepharose 6B on purified diamine oxidase from the serum of a man pretreated with heparin (Tufvesson, 1978b). However, a similar technique used on the serum diamine oxidase and amniotic fluid diamine oxidase of pregnant women revealed the presence of two active fractions in both which had native molecular weights of 245,000 and 485,000 (Tufvesson, 1978a, 1978b). Treatment of highly purified diamine oxidase from amniotic fluid with 1% SDS and 1% 2-mercaptoethanol followed by SDS-PAGE revealed the presence of a subunit with an apparent molecular weight of 100,000 dalton (Tufvesson, 1978a). Significantly, Tufvesson determined this apparent molecular weight in the presence of borate buffer which might be expected to lead to an overestimation of the subunit molecular weight. It is important to note here that Tufvesson did not determine the subunit molecular weight of his serum diamine oxidase preparation. Instead, he assumed that the pregnancy serum and amniotic fluid enzymes were identical presumably because of their identical apparent native molecular weights.
The isoelectric point of purified human amniotic fluid diamine oxidase was determined by measuring the activity of the bands which separated in two active fractions with pI values of 4.0 and 5.8. In considering the results for the isoelectric pH obtained for human placental diamine oxidase, Lin (1981) observed that the purified enzyme separated into 5 major bands and several diffuse bands, with the isoelectric pH of the major bands ranging from 5.3-6.6. In comparison, Crabbe (1976) determined the isoelectric point for human placental diamine oxidase to be 6.5. Thus, there is considerable confusion concerning the apparent isoelectric points of the various forms of diamine oxidase. It is highly likely that the observed isoelectric points are dependant on the method of purification or other factors because of the conflicting results of Lin (1981) and Crabbe (1976).
In respect to the several forms of DAO discussed above, it will be realised that pregnancy greatly modifies all metabolic patterns of histamine, putrescine and polyamines. Histamine and putrescine are produced in large amounts by the fetus. The level of DAO rises in parallel with the levels of histamine and putrescine during pregnancy (Zeller above). During a normal pregnancy the level of DAO in maternal plasma begins to rise from the second to third month reaching a maximum by the fifth to seventh month and remains at this level until parturition. The level of DAO in plasma falls quite rapidly after parturition (Lorenz, Kusche, 1970). The major function of DAO during pregnancy appears to be to ensure that the levels of the biogenic amines in the placental microcirculation do not become elevated and thus toxic to the developing fetus (Buffoni, 1966).
Premature rupture of the fetal membranes was observed in 6-16% of all pregnancies (Swartz, Napolitani, et al 1969), while another study found the rupture of fetal membranes in 3-14% of all pregnancies was not followed by labour (Larsen, 1979). On rupture of the membranes, Eastman & Hellman (1961) observed that spontaneous labour followed within 24 hours in 80% of patients, with 10% remaining undelivered after a period of 48 hours. The perinatal mortality doubles after a latent period of 24 hours and again after 48 hours (Overstreet and Romney, 1966). Delayed onset of labour carries with it an increased risk of maternal and perinatal infection and mortality (Eastman & Hellman; Kapeller-Adler & Macfarlane; Overstreet above), therefore the accurate detection of ruptured membranes is an important diagnostic aid.
In the mature fetus therefore, early diagnosis of membrane rupture allows for expediting the delivery thereby reducing infection risk to mother and baby. In the premature infant, accurate diagnosis of membrane rupture is even more important particularly if labour needs to be induced. A reliable, easy method of diagnosis of membrane rupture would be clinically useful to the obstetrician and also cost saving by reducing the need for prolonged and unnecessary hospitalisation. The benefit to those patients resident in the country areas and facing transfer to a tertiary care centre for prolonged periods of hospitalisation away from home and family environment on suspicion of having premature rupture of the membranes would be considerable.
High concentrations of DAO found in amniotic fluid are almost equal to that of maternal blood plasma (Tornqvist, 1971). After rupture of the membranes, DAO is demonstrable in the vagina; wherefore in absence of vaginal bleeding or exudation the measurement of the DAO content of vaginal fluid has been shown to be a reliable method in evaluation of membrane rupture (Ahlmark, 1944); Elmfors, Tryding, et al (1974); Gahl, Kozina, et al (1982). Besides physical examination, current techniques for establishing the diagnosis of ruptured fetal membranes are (Elmfors, Tryding (1976))
1. Determination of the vaginal pH; PA0 2. Staining for fetal fat globules; PA0 3. Identification of fetal squamous cells or hairs; PA0 4. Examination for typical crystallisation of amniotic fluid (ferming). PA0 1. urine; PA0 2. meoonium; PA0 3. antiseptics; PA0 4. pregnancy serum; PA0 5. seminal fluid; PA0 6. haemoglobin
In accordance with Friedman and McElin (1976) any three of the abovementioned methods taken together provide an accuracy of 93% although false-positive and false-negative results are frequent with all laboratory techniques which are applied in diagnosing rupture of the membranes (Larsen above). Dyes and other chemicals have been injected into the amniotic fluid transabdominally and their appearance looked for in the vagina but these techniques are not without risk (Jimsenez-Balderaz E. A. Bol. Med. Hosp. Infant Mex. 1984, 41, P341-4). Measurement of amniotic fluid volume by ultrasound before and after bed rest may be helpful but is not specific (Rudd above).
Measurements of the protein alpha-fetoprotein and the hormones prolactin and human placental lactogen have been investigated by Huber, J. F. et al (Huber, J. F. et al, Are Vaginal Fluid Concentrations of Prolactin, alpha-Fetoprotein and Human Placental Lactogen Useful for Diagnosing Ruptured Membranes, Br. J. Obstet. Gynacol. 90, 1183-1185) but he found many positive tests in patients whose membranes were intact. This was due in part to the fact that these substances are present in maternal serum and therefore the test is positive when either serum or amniotic fluid or both are present in the vagina.
Rochelson B. C. (Rochelson, B. C., A rapid colorimetric AFP monoclonal antibody test for the diagnosis of preterm rupture of the membranes. Obstet, Gynaecol, 1987, 69, P163-6) has also found blood and serum to give false positive results with his test for the presence of alphafetaprotein in vaginal fluid). Elmfors et al (above), using the information of Tornqvist & Jonassen (above) developed a method for the diagnosis of ruptured membranes by measuring the DAO activity in vaginal fluid. DAO activity is present in amniotic fluid but normally absent from vaginal secretions, presenting a method for diagnosing rupture of the membrane (Elmfors, Tryding et al above; Gahl, Kozina, et al above). The most widely used method for collection of DAO activity in vaginal secretions is the use of blotting paper to collect the secretions and phosphate buffer to elute the enzyme (Elmfors, Tryding above). Wishart, Jenkins et al (1979) also supported the use of the procedure and found an assay to offer useful clinical information. The factors which suggest that the DAO assay may be used as an adjunct to conventional tests in the diagnosis of rupture of membranes have been summarised by Gahl et al (above). However, the major drawback of the assay is that it depends on the measurement of enzyme activity and can give false results in the presence of interfering substances such as:
These substances commonly contaminate the vagina therefore invalidating current methods which are limited by interfering substances, inadequate sensitivity and subjective interpretation of results.